Abstract: My goal is to develop state-of-art imaging technology that can measure protein complex formation and protein networks in a multiplexed fashion with spatial resolution beyond that of the optical microscopy. At present, a major limitation to clarifying the dynamics of a particular signaling cascade is the inability to visualize multiple (>4) proteins and their interactions simultaneously in real time in the living cell. This is due in part to the interference of spectrally similar species (including cellular autofluorescence) and the mismatch between the spatial resolution of the confocal microscope and the scale of protein interactions. Computational and experimental approaches can help to elucidate many of these interactions, but not all. Specialized microscopy methods have been developed to address some aspects of the problem, but to date, no technology has demonstrated true multiplexed (simultaneous, not sequential) detection of >4 proteins and their complex formation in living cells at spatial resolutions >100 nm. This type of detection is critical for unraveling protein interaction network details, and my proposed work will address that. Specifically, I will: 1) implement novel emission-scanning hyperspectral confocal microscopy hardware to collect information from large numbers of fluorescent species simultaneously at spatial resolution beyond that of the optical microscope. 2) develop corresponding algorithms to spectrally unmix the 6D (X, Y, Z, excitation ?, emission ?, and time) images and provide accurate measurements of fluorophore concentrations even in the presence of energy transfer. This creative approach alleviates limitations of existing multicolor technology by extending my expertise in livecell hyperspectral imaging technology into the "super-resolution" realm. Its success will be enabled by robust multivariate image analysis algorithms. This advance will have far-reaching impact in exploring signaling pathways and networks in biology and biomedicine. Public Health Relevance: The complex symphony of signaling networks still remains a mystery. This project will develop a novel technology to unravel the details of signaling protein networks and pathways with extreme accuracy and spatial resolution. This work is very relevant to Public Health because cell signaling cascades control and regulate response to disease or therapeutic countermeasures.